Method for evaluating cardiomyocytes using raman scattering

Abstract

Method for evaluating cardiomyocytes using Raman scattering: a Raman spectrum of cardiomyocytes artificially induced to differentiate from pluripotent stem cells is acquired, an intensity of Raman-scattered light for a protein containing at least one of heme b and heme c as a prosthetic group is acquired from the Raman spectrum, and a state of progress of maturation of the cardiomyocytes is evaluated on the basis of the intensity of the Raman-scattered light. Method for evaluating differentiation into cardiomyocytes using Raman scattering: cells which are pluripotent stem cells are artificially induced to differentiate into cardiomyocytes, a Raman spectrum of the cells induced to differentiate is acquired, an intensity of Raman-scattered light for at least one of heme b and heme c is acquired from the Raman spectrum, and a state of progress of differentiation of the cells into cardiomyocytes is evaluated on the basis of the intensity of the Raman-scattered light.

1 . A method for evaluating cardiomyocytes using Raman scattering, the method comprising:
 acquiring a Raman spectrum of cardiomyocytes artificially induced to differentiate from pluripotent stem cells;   acquiring an intensity of Raman-scattered light for a protein containing at least one of heme b and heme c as a prosthetic group from the Raman spectrum; and   evaluating a state of progress of maturation of the cardiomyocytes on the basis of the intensity of the Raman-scattered light.   
     
     
         2 . The method according to  claim 1 , wherein
 the protein containing at least one of heme b and heme c as a prosthetic group is reduced cytochrome c, and   the intensity of the Raman-scattered light, which is Raman-scattered light for the reduced cytochrome c is acquired, and a peak of the intensity is calculated on the basis of an intensity of Raman-scattered light at a Raman shift of 743 cm −1  to 755 cm −1  in wavenumber.   
     
     
         3 . The method according to  claim 1 , wherein
 the protein containing at least one of heme b and heme c as a prosthetic group is oxymyoglobin, and   an intensity of Raman-scattered light for the oxymyoglobin is acquired, and the intensity is calculated on the basis of at least an intensity of Raman-scattered light at a Raman shift of 565 cm −1  to 575 cm −1  in wavenumber.   
     
     
         4 . The method according to  claim 1 , wherein the Raman spectrum excited by light having a wavelength of 532 nm is used. 
     
     
         5 . The method according to  claim 1 , wherein
 an intensity of Raman-scattered light for lipid is further acquired from the Raman spectrum, and   a state of progress of maturation of cardiomyocytes is evaluated on the basis of the intensity of the Raman-scattered light for lipid.   
     
     
         6 . The method according to  claim 1 , wherein the pluripotent stem cells are human cells. 
     
     
         7 . The method according to  claim 6 , wherein the cardiomyocytes aggregate into a sheet-shaped tissue fragment. 
     
     
         8 . The method according to  claim 6 , wherein the cardiomyocytes aggregate around a core to form a cell mass. 
     
     
         9 . The method according to  claim 6 , wherein the cardiomyocytes form a spheroid with human cardiac fibroblast cells, human cardiac endothelial cells and human mesenchymal cells, or
 the cardiomyocytes form a spheroid with human cardiac fibroblast cells and human cardiac endothelial cells.   
     
     
         10 . The method according to  claim 6 , wherein the cardiomyocytes form a cardiac organoid. 
     
     
         11 . The method according to  claim 6 , further comprising, before acquiring the Raman spectrum, accelerating maturation of the cardiomyocytes by at least one of:
 application of a mechanical stimulus to the cardiomyocytes;   application of an electrical stimulus to the cardiomyocytes;   introduction of a gene to the cardiomyocytes;   co-culture of the cardiomyocytes with other cells; and   addition of an oxidizing substrate, a compound and a factor to a medium in which the cardiomyocytes are cultured.   
     
     
         12 . The method according to  claim 6 , wherein acquiring the Raman spectrum, acquiring the intensity of the Raman-scattered light and evaluating the state of progress of maturation is repeatedly applied to the same cardiomyocytes over time. 
     
     
         13 . The method according to  claim 6 , further comprising, before acquiring the Raman spectrum, culturing the cardiomyocytes in the presence of a drug, wherein
 the state of progress of maturation of the cardiomyocytes is evaluated to evaluate an effect of the drug on maturation of the cardiomyocytes.   
     
     
         14 . The method according to  claim 1 , wherein the pluripotent stem cells are one of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and embryonic germ cells (EGCs), and
 the embryonic stem cells (ESCs) include nuclear transfer embryonic stem cells (ntESCs).   
     
     
         15 . The method according to  claim 1 , wherein a peak of excitation light in the acquisition of the Raman spectrum is in a range of 400 nm to 600 nm. 
     
     
         16 . The method according to  claim 1 , wherein a temperature of the cardiomyocytes is decreased to 4° C. or lower in the acquisition of the Raman spectrum. 
     
     
         17 . A method for evaluating differentiation into cardiomyocytes using Raman scattering, the method comprising:
 artificially inducing cells, which are pluripotent stem cells, to differentiate into cardiomyocytes;   acquiring a Raman spectrum of the cells induced to differentiate;   acquiring an intensity of Raman-scattered light for at least one of heme b and heme c from the Raman spectrum; and   evaluating a state of progress of differentiation of the cells into cardiomyocytes on the basis of the intensity of the Raman-scattered light.